Histamine release in the prefrontal cortex excites fast-spiking interneurons while GABA released from the same axons inhibits pyramidal cells

We studied how histamine and GABA release from axons originating from the hypothalamic tuberomammillary nucleus (TMN) and projecting to the prefrontal cortex (PFC) influences circuit processing. We opto-stimulated histamine/GABA from genetically-defined TMN axons that express the histidine decarboxylase gene (TMNHDC axons). Whole-cell recordings from PFC neurons in layer 2/3 of prelimbic (PL), anterior cingulate (AC) and infralimbic (IL) regions were used to monitor excitability before and after opto-stimulated histamine/GABA release in male and female mice. We found that histamine-GABA release influences the PFC through actions on distinct neuronal types: the histamine stimulates fast-spiking interneurons; and the released GABA enhances tonic (extrasynaptic) inhibition on pyramidal cells (PyrNs). For fast spiking non-accommodating interneurons, histamine released from TMNHDC axons induced additive gain changes, which were blocked by histamine H1 and H2 receptor antagonists. The excitability of other fast-spiking interneurons in the PFC was not altered. In contrast, the GABA released from TMNHDC axons predominantly produced divisive gain changes in PyrNs, increasing their resting input conductance, and decreasing the slope of the input-output relationship. This inhibitory effect on PyrNs was not blocked by histamine receptor antagonists but was blocked by GABAA receptor antagonists. Across the adult lifespan (from 3 months to 18 months of age), the GABA released from TMNHDC axons in the PFC inhibited PyrN excitability significantly more in older mice. For individuals that maintain cognitive performance into later life, the increases in TMNHDC GABA modulation of PyrNs during ageing could enhance information processing and be an adaptive mechanism to buttress cognition.


Introduction
Histamine, a wake-specific neuromodulator, is produced by tuberomammillary nucleus (TMN) neurons of the posterior hypothalamus (Watanabe et al., 1983;Panula et al., 1984;Haas and Panula, 2003;Scammell et al., 2019;Yoshikawa et al., 2021) that send axons throughout the brain (Takagi et al., 1986;Airaksinen and Panula, 1988;Haas and Panula, 2003;Arrigoni and Fuller, 2021;Yoshikawa et al., 2021), and are defined by expression of the histidine decarboxylase (hdc) gene, which encodes the enzyme that synthesizes histamine (Joseph et al., 1990). Histamine is released from axonal varicosities (Takagi et al., 1986), and activates excitatory H1, H2 and inhibitory H3 metabotropic receptors (Haas and Panula, 2016). As histamine acts by volume/paracrine transmission, it influences multiple elements of the circuitry, fine tuning both inhibitory and excitatory transmission (Ellender et al., 2011;Bolam and Ellender, 2016). Increased firing of histamine neurons in the TMN is associated with the waking state, and antihistamines that cross the blood brain barrier cause drowsiness. Therefore, it is expected that histamine release within the neocortex will result in excitation of the cortical circuitry.
As well as causing fast (millisecond) synaptic inhibition through GABA A receptors, GABA can diffuse to extrasynaptic ionotropic GABA A receptors to produce a tonic shunting inhibition (Brickley et al., 1996;Brickley et al., 2001;Wall, 2002;Brickley and Mody, 2012;Lee and Maguire, 2014;Brickley et al., 2018). Knockdown of vgat in hdc cells enhanced activity of mice and reduced their sleep. In the visual cortex, light-evoked GABA release increased tonic inhibition onto pyramidal cells, which was diminished when vgat gene expression was knocked down (Yu et al., 2015). Therefore, our work has challenged the view that histaminergic neurons' influence on cortical circuitry will be purely excitatory.
Vgat expression in histaminergic cells has become controversial, however. Combined immunocytochemistry and in situ hybridization for HDC protein and vgat mRNA revealed little co-expression (Venner et al., 2019), but single-cell RNA seq studies of mouse TMN identified a subset (E5) of hdc cells with moderate expression of vgat (Mickelsen et al., 2020). This subset of cells, due to their long-range projections, could still have widespread influence. Therefore, in this study we have extended our analysis to the prefrontal cortex (PFC) and broadened our investigation to assay excitability changes in pyramidal cells and interneurons. We have used whole-cell recording techniques combined with optogenetics to describe how the gain of the input-output relationship is altered following GABA/histamine release from hdc-expressing TMN axons (TMN HDC axons). Importantly, we have now recorded over a much broader age range than in our previous study (Yu et al., 2015) to ask whether the changes in excitability associated with GABA/histamine release are uniform across the adult lifespan. For the first time, we show that GABA release is elicited from genetically defined histaminergic axons in the PFC of young and old mice (of both sexes) and that the GABA/histamine co-transmission works together to enhance pyramidal cell information transfer, especially in older animals.

Mice
All procedures were performed in accordance with the United Kingdom Home Office Animal Procedures Act (1986) and were approved by the Imperial College Ethical Review Committee. The strains of mice used were HDC-Cre (Hdc tm1.1(icre)WwiS/J , JAX stock 021198) (Zecharia et al., 2012) and Pv-Cre (Pvalb tm1(cre)Arbr/J , JAX stock 008069), kindly provided by S. Arber (Hippenmeyer et al., 2005). The HDC-Cre mouse line contains an ires-Cre cassette knocked-in to exon 12 of the hdc gene. The insertion is downstream of the stop codon for the hdc reading frame, and therefore the knock-in allele makes HDC and Cre proteins (Zecharia et al., 2012).
After each injection, pipettes were left undisturbed for 10 min to allow the viral solution to be absorbed by the tissue; thereafter, pipettes were removed slowly. After injection, craniotomies were sealed with Kwik-Cast (World Precision Instruments, USA), and the scalp was sutured with glue (Histoacryl, Braun, USA) or nonabsorbable nylon sutures (Ethilon, Ethicon, USA). Immediately after suturing, anaesthesia was antagonized via injection of a naloxone, flumazenil, and atipamezole mixture (1.2, 0.5, and 2.5 mg/kg, i.p.). Postoperative analgesia was provided via eutectic mixture of local anaesthetics (EMLA; 2.5% lidocaine and prilocaine) application to the sutured skin. Mice were placed in a heated recovery box under observation until they fully woke up from anaesthesia; thereafter, animals were returned to their home cage. Four to five weeks after the AAV injection, we prepared acute slice preparations containing the TMN area. If there was visible primary fluorescence in this area we also went on to slice the PFC. Initially, we also tested for the presence of ChR2 by patching the cells fluorescent TMN cells, and in current clamp mode and eliciting an inward current response with blue light stimulation of ChR2 to confirm the presence of functional ChR2. Slices from TMN injections that gave no visible primary expression were discarded or used as negative controls.
Optogenetics-A 470 nm collimated LED (M470L3-C1, Thorlabs) was used to illuminate the slice through the objective lens. The LED was driven by an LED-driver (LEDD1B, Thorlabs) which was controlled by a National Instruments digitization board (NI-DAQmx, PCI-6052E; National Instruments, Austin, Texas). The output of the LED was measured with a power meter (PM100D, Thorlabs) positioned below the objective lens and the power output and membrane conductance or voltage changes were aligned for analysis. The optical power emitted through our 63X water immersion lens increased linearly to a maximum power of 70 mW/mm 2 at our chosen light stimuli, giving rise to a transient response that peaked at 40 mW/mm 2 , with a 10%-90% rise time of 0.73 ms and a decay constant of 9.65 ms.
Data analysis-For all recorded cells, total membrane capacitance (C m ) was calculated in voltage-clamp configuration from Cm = Q/ΔV, where Q was the charge transfer during a hyperpolarizing 10 mV step of the command voltage (ΔV). The total membrane conductance (G m ) was calculated from G m = I ss /ΔV, where I ss was the average steady-state current during the ΔV. The electrode-to-cell series resistance (Rs) was calculated from the relationship Rs = ΔV/I p , where I p was the peak of the capacitive current transient and recordings were excluded if Rs increased by >30%.
Experimental Design and Statistical Analysis-To test for statistical significance between single-cell properties across our broad range of ages, we first applied the Mann-Whitney non-parametric test. We then applied the Benjamini-Hochberg (B-H) correction at p= 0.05 and 0.01 values to control for multiple comparisons. As we were looking for changes in many biophysical properties we chose the B-H correction procedure as it is less sensitive than the Bonferroni procedure to decisions about the identity of a "family" of tests. Briefly, the B-H procedure ranks individual p-values and then compares individual p-values to their B-H critical value, (i/m)*Q, where i is the rank, m is the total number of tests, and Q is the false discovery rate. The largest p-value that has p < (i/m)*Q is considered significant.

Results
Four to five weeks prior to whole-cell recording experiments, an AAV containing a flex-ChR2-EYFP transgene was delivered into the TMN of HDC-Cre mice ( Figure 1A). The expression of ChR2-EYFP was verified in the TMN ( Figure 1B) and, like our previous reports, we found that ChR2-EYFP expression as restricted to a subset of hdcexpressing neurons of the TMN ( Figure C). Whole-cell recordings were then made from fast-spiking interneurons (FS-INs) and pyramidal neurons (PyrNs) within layer 2/3 of the prelimbic (PL), anterior cingulate (AC) and infralimbic (IL) regions of the PFC ( Figure  1D) Following these recordings, the presence of fluorescently labelled TMN HDC axons was observed in the PFC ( Figure 1E). 3D reconstructions of biocytin filled cells in the PFC demonstrated how these fluorescently labelled TMN HDC axons rarely make close appositions (< 1μm) with PyrN dendrites ( Figure 1F), as expected from previous studies (Takagi et al., 1986). Pyramidal neurons (PyrNs), fast-spiking accommodating (FS-IN a ) and fast-spiking non-accommodating interneurons (FS-IN na ) in layer 2/3 were selected according to their location, soma shape, and electrophysiological features. Most strikingly, PyrNs exhibited evoked AP firing rates of below 20 s -1 , with an average maximum AP rate of 11.4 ± 0.5 s -1 (n = 19), whereas the evoked firing rates of FS-INs was between 20 s -1 to 90 s -1 with an average maximum AP rate of 46.6 ± 3.8 s -1 (n = 19) ( Figure  1G). To further confirm the identity of FS-INs, we patched GFP-fluorescent cells from PV-Cre mice following AAV-flex-GFP injection into the PFC (n = 4). As expected, AP characteristics for the parvalbumin-expressing cells were like those observed in the FS-IN group. A further breakdown of the FS-IN group was based on differences in the inter spike intervals (ISIs) of FS-INs (see Figure 1H). Cells with stable ISIs were classified as non-  (Feldmeyer et al., 2018). Based upon firing properties alone, the irregular spiking population (FS-IN a ) are likely to represent the somatostatinpositive Martinotti and non-Martinotti cells that target inhibition to the basal and apical dendrites of PyrNs (Feldmeyer et al., 2018). However, additional morphological and immunohistochemical analysis would be required to confirm this classification (Krimer et al., 2005).

Optogenetic stimulation of TMN HDC axons alters excitability in the PFC
During whole-cell recording, TMN HDC fibres in the PFC slices were activated with 1 ms duration blue light flashes delivered at a rate of 5 s-1 every 0.4 seconds for a total stimulation period of 4 minutes (Yu et al., 2015). This protocol was chosen to mimic the short 5 s -1 AP bursts that have been observed for several minutes when recording from wake-active TMN neurons during attentive waking in mice (Takahashi et al., 2006). In the example shown in Figure 2A, simultaneous whole-cell recordings were made from a FS-IN na and a PyrN. In PyrNs, a reduction in AP firing rates following TMN HDC axon stimulation was obvious from the input-output (I-O) relationships constructed from average AP rates. As shown in Figure 2B,C, the I-O relationships for data obtained from an individual PyrN ( Figure 2B) and a FS-IN na ( Figure 2C) could be well described by a sigmoidal function, both before and after optogenetic stimulation. Results from the fits obtained from 19 PyrNs were pooled, and the average I-O relationship before and after optogenetic stimulation of TMN HDC axons was constructed ( Figure 2D). This analysis demonstrated that the maximum AP firing rate was reduced from 12.7 ± 1.6 s -1 to 3.9 ± 5.3 s -1 following stimulation of TMN HDC axons (Wilcoxon signed rank test, p=0.0002). The slope of the average I-O relationship significantly reduced from 7.6 ± 0.9 to 2.4 ± 0.7 (Wilcoxon signed rank test, p=0.0004) with no significant reduction in the current required to reach 50% of the maximum AP rate (50.4 ± 36.2 pA before stimulation and 49.9 ± 18.9 pA after stimulation; Wilcoxon signed rank test, p=0.7). Transforming the average, I-O relationship obtained under control conditions with a purely divisive function indicates that an additive gain change mechanism may make a minor contribution to changes in PyrN excitability. Specifically, there was a small leftward shift in the current required to reach AP threshold that was not predicted from a purely divisive model ( Figure 2D). However, the most striking aspect of the changes in the I-O relationships constructed from PyrN recordings were the divisive effects observed following TMN HDC axon stimulation.
The average firing rates of the FS-IN na population (n=8) significantly increased following optogenetic stimulation of TMN HDC axons ( Figure 2E). The change in the average I-O relationship of the FS-IN na population was consistent with an additive gain change mechanism ( Figure 2E). The maximum AP firing rate increased from 39.8 ± 3.8 s -1 to 44.8 ± 3.8 s-1 (Wilcoxon signed rank test, p=0.008) with 50% excitation point shifting from 82.4 ± 17.8 pA to 70.0 ± 16.2 pA (Wilcoxon singed rank test, p=0.5). However, there was also a small but significant reduction in the slope of the input-output relationship from 0.3 ± 0.2 to 0.2 ± 0.1 (Wilcoxon signed rank test, p=0.008). Therefore, like the situation for PyrNs, analysis of I-O relationships suggests that two types of gain mechanism could operate within the FS-IN na population. However, transforming fits from the FS-IN na population solely with an additive function predicted well the changes observed in the I-O relationship.
In contrast to the clear changes observed for PyrNs and FS-IN na s, the excitability of the FS-IN a population was unchanged following optogenetic stimulation of TMN HDC axons in the PFC (n=4), such that the maximum AP rate was 30.4 ± 1.8 s-1 before stimulation and 29.2 ± 2.2 s-1 after. There was also no significant change in the slope of the I-O relationship (6.8 ± 1.4 versus 6.4 ± 1.4) or the current required to reach 50% of the maximum AP rate (28.2 ± 7.1 pA versus 32.9 ± 11.1 pA) in the FS-IN a population ( Figure 2F).
Consistent with an additive gain change mechanism, neither the resting membrane potential or the input conductance of the FS-IN na population was altered following stimulation of TMN HDC axons (see figure 2G). The resting membrane potential of the PyrN population was also not altered during the tonic shunting inhibition associated with this divisive gain change. However, consistent with a divisive gain change mechanism, there was a significant 20% increase in membrane conductance in PyrNs following optogenetic stimulation that was consistent with a shunting inhibition (see Figure 5E) (n pyr = 22, p < 0.02 using paired sample Wilcoxon Signed rank test). The divisive gain change observed in PyrNs is consistent with GABA binding to extrasynaptic GABA A receptors, whereas the additive gain change associated with the FS-IN na population is more likely to be due to modulation of excitability following the action of histamine on G-protein coupled receptors. To test this hypothesis, we next undertook some simple pharmacological experiments, during which the time course of the gain change was also examined.
The PyrN divisive gain change involves GABA A receptors-To compare the time course associated with changes in PyrN excitability the average AP rate was calculated during the entire depolarizing current injection protocol ( Figure 3A). The subthreshold membrane voltage appeared more linear following TMN HDC axon activation and in 10 out of 22 PyrNs tested, no APs could be elicited following optogenetic stimulation. However, with larger depolarising currents, AP firing was possible in these PyrNs (data not shown). Simple Boltzmann functions fitted to the time course plots demonstrated that the reduction in PyrN firing was apparent at 2-3 minutes following TMN HDC axon activation ( Figure 3B). Analysis of all fits demonstrated that the average AP rate reduced significantly from 10.7 ± 0.8 s -1 to 4.5 ± 0.9 s -1 following optogenetic stimulation (n=22) and the average change in the AP rate for this group was -65.7% ± 8.4%. Several negative control experiments were conducted with acute PFC slices from HDC-Cre mice (n = 8) which had not received AAV-flex-ChR2 injections into the TMN. In these experiments, the optogenetic protocol resulted in a -15.9% ± 7.0% change in AP firing rates in PyrNs that was not significant (n = 8, p < 0.06, Wilcoxon Signed Ranks Test). There was also no change in resting membrane potential or membrane conductance in these PyrNs. As a further control, the addition of the GABAA receptor antagonist SR95531 blocked the actions of TMN HDC axon stimulation, resulting in only a small reduction in AP firing rates in PyrNs of -7.7% ± 10% following Europe PMC Funders Author Manuscripts optogenetic stimulation that was also not significant (n H1,H2 = 5, n GA B A-A = 5, p < 0.029 by Mann Whitney).
Pharmacological experiments were undertaken to directly address the involvement of H1/H2 receptors in the gain change. The optogenetic experiments described above were repeated in the presence of H1 (pyrilamine) and H2 (ranitidine) receptor antagonists. Immediately after the TMN HDC axon stimulation protocol, a larger depolarising current was required to elicit APs in PyrNs ( Figure 3C) and, on average, the AP firing rate in PyrNs decreased by -70.0% ± 15.8% in the presence of H1/H2 antagonists (n=5, p < 0.032, Wilcoxon Signed Ranks Test). In the example shown in Figure 3C, the average AP rate was 6.4 s -1 prior to blue light stimulation but was zero following stimulation. Therefore, in this PyrN the cell excitability was considered to have been reduced by -100% (see Figure 3D). Clear changes in the subthreshold voltage behaviour of this PyrN was still observed following TMN HDC axon stimulation. The net inhibition of PyrNs observed following TMN HDC axon stimulation in the PFC was -65.7% ± 8.4% (n=22) in control conditions compared to -70.0% ± 15.8% (n=5) in the presence of H1/H2 antagonists. A similar fast time course for PyrN AP firing rate changes were also observed in the presence of the H1/H2 blockers.

The FS-IN na additive gain change involves histamine receptors
Once again, the excitability of the FS-IN a population was unchanged following optogenetic stimulation of TMN HDC axons in the PFC but, the FS-IN na population became more excitable ( Figure 4A). In this example the average AP rate increased from 10.6 s -1 to 171.7 s -1 and this additive gain change was not associated with any change in the resting input conductance as evidenced by the linear subthreshold IV relationship before and after stimulation of TMN HDC axons. The time course plots demonstrated that the enhancement of FS-IN na firing was noticeably delayed relative to the end of the blue light stimulation ( Figure 4B) with the increase in AP rate occurring 10 minutes after the end of blue light stimulation at a much slower rate than that apparent for the reduction in PyrN excitability. This slow time course of the increase in AP firing could reflect a more gradual increase in histamine concentrations in the extracellular space or a mechanism involving modulation by G-protein-coupled receptors such as the H1/H2 receptors, whereas the rapid time course of the reduction in AP firing in PyrNs is consistent with the rapid binding of GABA to high-affinity ligand-gated ion channels. To examine the contribution of H1/H2 receptors in the additive gain change the optogenetic experiments described above were repeated in the presence of H1 (pyrilamine) and H2 (ranitidine) receptor antagonists. With histamine receptors blocked there was no change in the average AP rate after the TMN HDC axon stimulation s ( Figure 4C) and, on average, the AP firing rate in the FS-IN na population was not increased in the presence of H1/H2 antagonists ( Figure 4D).

Age differences associated with TMN HDC modulation of the PFC
We next explored the age-dependence of TMN HDC modulation of the PFC. First, we examined if there were any age or sex related differences in the resting input conductance of PyrNs. As shown Figure 5A, the resting input conductance of PyrNs was similar across the adult lifespan and this parameter was not influenced by an animal's sex. A linear regression analysis of this data (male & female) resulted in a negative slope and a Pearson's correlation coefficient or r 2 of -0.03, demonstrating how variability in the resting input conductance was not significantly influenced by the age of the animal (ANOVA, p=0.72). Across the entire lifespan, the resting input conductance of the PyrNs recorded from male mice was 4.44 ± 0.36 nS (n=55) compared to 5.44 ± 0.47 nS (n=46) for female mice. Much of the variability observed for the resting input conductance in the PyrN population (4.90 ± 0.36 nS, n = 101) was expected to be explained by variability in cell size as determined from the membrane capacitance (39.1 ± 3.6 pF). Therefore, a least square regression analysis ( Figure 5B) of the normalised data demonstrates a slope of +0.34 with an r 2 value of 0.37 indicating that nearly 40% of the variability in the input conductance (PC2) can be explained by differences in cell size (PC1). Hypothesis testing using ANOVA demonstrated how the slope of this relationship was significantly greater than zero (p=0.0003). Next, we looked for changes in the response of PyrNs to GABA/histamine release from TMNHDC axons across the adult lifespan by plotting the change in AP rate as a function of age for PyNs ( Figure 5C, n = 20).
A simple regression analysis of this data demonstrated that the magnitude of this inhibition increased as a function of age with a slope of -0.03 and an r 2 value of -0,45, indicating that nearly 50% of the variability in TMN HDC modulation can be explained by age. Furthermore, ANOVA analysis demonstrated that at the p<0.05 level, the slope of this relationship was significantly different from zero (p=0.045). Therefore, TMN HDC axon modulation of PyrN excitability is maintained throughout the adult lifespan with a tendency to increase with age.
We repeated this analysis for the two interneuron populations we have recorded in the mouse PFC. The resting input conductance of all FS-INs (n = 23 recordings) was not related to the age of the mouse ( Figure 5D) with no correlation observed in the data (r 2 = 0.02, ANOVA, p=0.58). Although the number of recordings in each group was small, the resting input conductance of FS-IN na and FS-IN a populations were similar in magnitude and did not appear to be influenced by sex or age. The enhancement of FS-IN na excitability was maintained across adult life, but the weak correlation (r 2 = 0.19) we observed within this smaller dataset (n = 8) was not significant ( Figure 5E). As expected, no change in the excitability of the FS-IN a population (n = 5) was observed at any of the ages examined in this study ( Figure 5E).
Consistent with modulation of a tonic GABA A receptor mediated conductance following GABA release from TMN HDC axons, we observed a clear enhancement of the resting input conductance in PyrN that followed the same time course as the reduction in AP firing (data not shown). Following TMN HDC activation, the input conductance increased from 6.52 ± 2.16 nS to 11.85 ± 1.65 nS ( Figure 5F). In contrast, no significant change was observed for the FS-IN na population with a resting input conductance of 2.79 ± 0.35 nS before and 3.07 ± 0.48 nS after stimulation (Wilcoxon Signed Rank Test, p=0.1). The increase in the input conductance of PyrNs correlated with the magnitude of the reduction in AP firing, as shown by the linear regression analysis of the normalised data ( Figure 5G) with an r 2 value for this fit of -0.71 with a slope that was significantly different from zero (ANOVA, p=0.0004). Therefore, the age-related changes we observe following TMN HDC modulation of AP firing can be explained by enhanced modulation of the input conductance.

Discussion
This study demonstrates how histamine/GABA released from TMNHDC axons alters AP firing within the mouse PFC in a cell-and age-specific manner. We recorded from three distinct classes of PFC neuron in prelimbic (PL), anterior cingulate (AC) and infralimbic (IL) regions of the PFC: layer 2/3 pyramidal neurons (PyrN), non-accommodating fastspiking interneurons (FS-INna) and accommodating fast-spiking interneurons (FS-INa). Each celltype responded differently to optogenetic stimulation of TMNHDC axons. The AP firing of the FS-IN a population was unaltered while the FS-IN na population (putative parvalbumin-positive cells) was excited and the PyrN population was robustly inhibited (see conceptual summary Figure 6). Pharmacological experiments confirmed that histamine release was responsible for FS-IN na excitation, whereas the GABA release from THN HDC axons was responsible for inhibition of PyrNs. By making recordings from mice over the adult lifespan (3-27 months postnatal), we found that shunting inhibition produced by GABA modulation was more pronounced in the adult and was sustained into older age.
Although the net effect of the wake-promoting histamine system in the brain is excitatory at the behavioural level (Haas and Panula, 2003;Scammell et al., 2019;, in the case of the neocortical network we propose that the histamine-GABA system influences the circuit's requirement for AP precision. On the one hand, we find that histamine stimulates a subset of fast-spiking GABAergic interneurons that will increase phasic inhibition onto PyrNs to enhance precision of PyrNs and expand their dynamic range to enhance synchrony in the PFC network (Krimer et al., 2005;Feldmeyer et al., 2018). On the other hand, the raised ambient GABA levels produced following TMN HDC axon GABA release will speed up the membrane time constant of PyrNs by enhancing the resting input conductance following extra synaptic GABAA receptor activation and, therefore, will also enhance coincidence detection, such that more closely timed EPSP inputs will be required to elicit APs (Brickley and Mody, 2012;Wlodarczyk et al., 2013;Sylantyev et al., 2020). By limiting the window of coincidence detection, tonic inhibition can promote conditions that enhance cognition. Moreover, we propose that the additive gain changes associated with the FS-IN na population of the PFC will further promote synchrony of the network during wakefulness.
Previously, gain changes within neocortical circuits were studied in relation to the action of neuromodulators such as serotonin and acetylcholine on interneurons (Ferguson and Cardin, 2020). However, this is different to the mechanism we propose, whereby GABA release from TMN HDC axons is directly responsible for the generation of a shunting inhibition ( Figure 6). Within minutes of TMN HDC opto-activation, the PyrN population experienced a dramatic reduction in the slope of the input-output relationship and a reduction in the maximum AP firing rate. As expected, this divisive gain change was associated with a significant 20% increase in the input conductance, with no change in the RMP, a characteristic of a tonic shunting inhibition mediated by extrasynaptic GABA A receptors (Mitchell and Silver, 2003). Therefore, GABA diffusion through the extracellular space is sufficiently unhindered to enable rapid coupling between the GABA released from TMN HDC axons and alterations in the gain of the surrounding PyrNs. This is similar to the mode of action reported in other cortical and striatal regions following TMN HDC activation (Yu et al., 2015), and consistent with observations following the knockout of high-affinity δ subunit-containing GABA A receptors (Abdurakhmanova et al., 2020). The lack of a shunting inhibition observed in FS-IN na and FS-IN a populations would suggest that the change in GABA levels produced following release from TMN HDC axons was insufficient to activate extrasynaptic GABA A receptors on these cells.
We have investigated the consequence of TMN HDC activation for local interneuron excitability and observed an increase in the gain of these neurons, but this gain change was predominantly additive, and the time course of the response was much slower than that observed for PyrNs. In contrast to the effects of TMN HDC activation on PyrNs, the additive gain change observed for the FS-IN na population was due to histamine's actions on H1/H2 receptors. The delayed increase in FS-IN na excitability is consistent with the reduction of an outward potassium conductance that was reported in hippocampal interneurons following H2 receptor activation and subsequent coupling to adenyl cyclase pathways (Atzori et al., 2000). The delayed nature of this excitability change is also consistent with the role of histamine release from TMN axons in maintaining wakefulness more than switching behavioural states (Takahashi et al., 2006).4 Moreover, the slow time course of the histamine effects on FS-IN na populations suggest that these changes will be sustained compared to the shunting inhibition observed for PyrNs. Future studies are clearly required to understand the interactions more fully between the actions of histamine and GABA in the PFC. However, what is clear from our data, is that the divisive gain change that results from the direct actions of TMN HDC axon-released GABA on extrasynaptic GABAA receptors dominates in the PFC, as this still occurrs in the presence of H1/H2 receptor blockers when the excitability of surrounding interneurons was not altered. This result demonstrates that the shunting inhibition is largely due to release of GABA from the TMN HDC axons and not greatly influenced by increased GABA release from local interneurons.
Human brain imaging studies have reported lower levels of variability in the resting state BOLD signal in the PFC of individuals in later life (Nomi et al., 2017), and this reduction in signal variability has been associated with overall decreased GABA levels in the cortex (Porges et al., 2021). In older humans, positive allosteric modulation of γ2 subunit-containing GABAA receptors, for example with lorazepam, may protect against cognitive decline (Lalwani et al., 2021). Tonic inhibition can come from GABA-activation of both γ2 and δ subunit-containing receptors, but only γ2 subunit-containing GABAA receptor mediated responses can be allosterically enhanced by benzodiazepines. Similarly, for individuals that maintain cognitive performance with aging, the increases in TMN HDC GABA modulation of PyrNs during ageing may be an adaptive mechanism to buttress cognition.
Finally, we note that our present and previous results on GABA-histamine corelease do differ from results reported in another study (Venner et al., 2019). In general, most findings, whether pharmacological or genetic, support the role of histamine in promoting wakefulness, although, as usually found, permanent gene or cell knockouts/lesions tend to give different results from reversible pharmacology or knockdowns: for example, hdc gene knockouts, and hdc cell lesions induce sleep-wake fragmentation (insomnia), but do not affect overall levels of sleep-wake (Parmentier et al., 2002;Yu et al., 2019); on the Lucaci et al. Page 11 other hand, histamine H1 receptor antagonists, chemogenetic or opto-inhibition inhibition of histamine neurons induces NREM-like sleep (Fujita et al., 2017;Yu et al., 2019;Naganuma et al., 2021;Yoshikawa et al., 2021). We have previously explored how levels of arousal are altered following chemogenetic manipulation of this TMN HDC projection (Yu et al., 2015), and found that activation of the TMN HDC projection increased motor activity, and that genetic knockdown of vgat from this pathway sustains wakefulness. In contrast, Venner et al. 2019 reported that chemogenetic manipulation of hdc cells did not affect sleep-wake behaviour and found no role for GABA in wakefulness (Venner et al., 2019;Arrigoni and Fuller, 2021). However, the Cre line used by Venner et al does not target all hdc-expressing neurons. For our mechanism of GABA release, we propose the mechanism is via VGAT (Yu et al., 2015), but in any case, GABA can also be transported into synaptic vesicles by the vesicular monoamine transporter VMAT (Tritsch et al., 2012), whose gene is strongly expressed in histaminergic cells (Mickelsen et al., 2020). Venner et al. 2019 did not provide electrophysiological evidence to support their contention that GABA was not released from TMN HDC axons in the neocortex, although they found using slice electrophysiology that histamine axons projecting to the preoptic hypothalamus did not release GABA. The best explanation for the disparities between studies are that the various subtypes of histamine neuron identified by RNAseq studies differ in their ability to release GABA (Mickelsen et al., 2020).
In summary, we present evidence that histamine released from TMN HDC axons is responsible for additive gain change within specific interneuron populations of the adult PFC. We speculate in Figure 6, that these changes in FS-IN na excitability will lead to increased synchronisation of cortical circuitry of the type (high frequency) associated with the awake brain (Yu et al., 2015). In contrast, the GABA released from these same TMN HDC axons leads to a divisive gain change that will broaden the dynamic range of PyrNs (see Figure 6) a feature that will lead to greater computational flexibility within the PFC. We have also shown that the shunting inhibition associated with GABA release increases with age and, it is intriguing to speculate that enhancement of this feature of TMN HDC modulation could be a compensation for age-related declines in global GABA levels observed within the PFC (Porges et al., 2021). Enhanced GABA modulation from TMN HDC axons could protect against reduced cognitive flexibility that is a feature of the ageing process while histamine release will support arousal due to modulation of local interneurons.

Significance statement
The hypothalamus controls arousal state by releasing chemical neurotransmitters throughout the brain to modulate neuronal excitability. Evidence is emerging that the release of multiple types of neurotransmitters may have opposing actions on neuronal populations in key cortical regions. This study demonstrates for the first time that the neurotransmitters histamine and GABA are released in the prefrontal cortex from axons originating from the tuberomammillary nucleus (TMN) of the hypothalamus. This work demonstrates how hypothalamic modulation of neuronal excitability is maintained throughout adult life, highlighting an unexpected aspect of the ageing process that may help maintain cognitive abilities.  illustrations were adapted from the Allen Brain Mouse Atlas. Bright field images were taken during whole cell recording and the fluorescent cell was recorded from the Parv-Cre mouse following flexed-GFP delivery into the PFC. E, Microscope images taken from the PFC region where recordings were made, and blue light was delivered. The left-hand bright field image demonstrates the location of the biocytin filled PFC neuron and the epifluorescent image on the right illustrates the presence of ChR2-EYFP labelled axon terminals in this region of the PFC. F, PFC neurons were reconstructed following confocal microscopy to reveal the proximity of the TMN axons relative to the recorded neuron. As expected, the ChR2-EYFP labelled axon terminals were never observed close enough (<1μm)  The solid black line is the average fit with the grey shaded area showing the 95% confidence limits for the fits obtained before stimulation. The blue solid line is the average fit and the shaded blue region is the 95% confidence limits for fits obtained after optogenetic stimulation. The dashed lines demonstrate the predictions when assuming a purely divisive gain change mechanism in PyrNs. E, Comparison of the average fits obtained from the input-output relationships constructed from the FS-IN na population (n=8) before and after optogenetic stimulation of the TMN HDC axon terminals in the PFC. In contrast to the data from PyrNs, an additive gain-change mechanism was solely responsible for the increased firing observed in the FS-IN na population. F, Comparison of the average fits obtained from the input-output relationships constructed from the FS-IN a population (n=4) before and after optogenetic stimulation of the TMN HDC axon terminals in the PFC. Note the lack of any change in the FS-IN a population. G, Scatter plots for the RMP recorded before and after optogenetic stimulation in PyrNs and FS-INs with the results of linear region analysis. The all-point histograms constructed before and after optogenetic stimulation further demonstrates how the RMP does not alter. The straight lines are the results of linear regression analysis with the 95% confidence limits shown in grey shaded areas.  time course of changes in average AP rate were described using a Boltzmann function to quantify the rate as well as the magnitude of the change in excitability. The shaded area indicates the 95% confidence limits for these fits. C, same conventions as A but the data was obtained from PyrNs in the presence of H1/H2 antagonists. Note in C how the subthreshold response of PyrNs becomes more linear following optogenetic stimulation of TMN HDC axons. D, combined scatter and violin plots illustrating the change in AP rate in control conditions with expression of ChR2 in TMN HDC axons (black symbols), with GABA A receptors blocked (dark grey symbols), and with H1/H2 blockers (red symbols). The final plot illustrates the results of a negative control experiment performed in the absence of ChR2 in TMN HDC terminals (light grey symbols).  A, Scatter plot of the resting input conductance taken from all PyrNs recorded from mice aged 3 months to 27 months. This data is separated into male (blue circles) and female (pink circles). The solid line is the result of linear regression analysis to the combined male and female data demonstrating a lack of change in input conductance across the adult lifespan. The grey shaded area shows the 95% confidence level for this fit and the r 2 value of -0.03 indicates how little of the variability in resting excitability can be explained by age. B, Normalised data comparing the relationship between the membrane capacitance (PC1) and the input conductance (PC2) for all PyrNs recorded across all ages. The linear regression analysis demonstrates that there is a relationship between these two parameters. C, the changes in AP rate obtained from PyrN recordings are plotted in relation to the animal's age. The results of linear regression analysis are superimposed on this scatter plot demonstrating Lucaci et al. Page 25 a relationship between the strength of the inhibition and the age of the animal. D, Scatter plot of data from FS-IN cells. Measurements from FS-IN a and FS-IN na cells have been separated into male and female but once again the linear region indicates that the resting excitability of FS-INs does not vary across the adult lifespan. E, the changes in AP rate obtained from interneuron recordings are plotted in relation to the animal's age. The results of linear regression analysis are superimposed on scatter plots obtained for the FS-IN na and the FS-IN a populations. F, Scatter plots of the resting input conductance measured before and after TMN HDC optogenetic stimulation. The dashed line has a slope of 1 to illustrate the enhancement of the input conductance that is apparent for the PyrN recordings but not the FS-IN na population. G, The change in AP rate data shown in panel C was replotted in relation to the change in input conductance for each PyrN. Once again the results of linear regression analysis are superimposed on this scatter plot. The significant negative correlation (p=0.0004) shows that larger increases in input conductance are associated with greater reduction in AP rate within the PyrN population.